The present invention relates to a method and apparatus for measuring and calibrating the measurement of a liquid volume. The liquid volume can be delivered from one or a multiplicity of orifices or tips simultaneously or sequentially. Also described are a test methodology, an algorithm for calculating results, a system of liquid reagents, a method of calibrating a photometric reader, and software that coordinates, controls and carries out these activities.
Many analysis methods used in biology, chemistry, biotechnology, pharmaceutical and other research laboratories require accurate measurement and/or calibration of small volumes of liquids. These small volumes can range from nanoliters to milliliters. In one application, small volumes of liquid are dispensed from liquid delivery devices comprising a single delivery orifice or multiple orifices configured to deliver liquid simultaneously or sequentially. Specific examples include handheld multichannel pipettes, configured to deliver 8 or 12 channels at a time, and automated delivery equipment configured to deliver 96 or 384 channels at one time. In other applications, there is measured a precise amount of liquid contained by a small volume vessel.
For liquid delivery devices, the delivery must be both accurate and precise. At any given time the delivery device may not be functioning within the requirements of the process or the specifications of the manufacturer. For this reason it is necessarily to periodically calibrate the delivery equipment to ensure its correct operation and the integrity of the analysis.
Multi-channel delivery devices are typically used to expedite the analysis or processing of many samples at once, or to analyze one sample for many different attributes at once. In order to assure the integrity of the multi-analysis process, the equipment must be functioning correctly at the time of the analysis. Existing calibration methods have various limitations that prevent timely, convenient, accurate and/or precise calibration activities, thus bringing into question the results of the analysis. Several of these existing calibration methods are described below.
In the gravimetric method, a liquid volume is determined by weight. After initially weighing a receiving tube, the liquid volume is delivered into the tube. The tube is reweighed and the weight gain of the filled tube leads to calculation of the liquid volume, after correction for the density of the fluid, the loss of fluid due to evaporation during the procedure, and the buoyancy of air. The gravimetric method, however, is extremely time consuming, particularly for calibrating multiple liquid volumes as done with multi-channel liquid delivery devices where hundreds of liquid deliveries occur simultaneously. In the case of small delivery volumes, the errors introduced into the weighing by vibration, draft, static electricity, and/or evaporation cause this method to be of questionable utility or validity.
The gravimetric method can be applied to multiple volumes by the use of microtiter plates as the receiving vessel. Microtiter plates are rectangular molded plastic plates having a multiplicity of small cavities or wells to receive the liquid being delivered. Exemplary microtiter plates have 96 or 384 wells. In this method, the amount of liquid delivered to the wells is not measured individually. A complete microtiter plate is weighed without the liquid, the wells are filled and the plate is then re-weighed. The resulting weight gain is converted to liquid volume by accounting for liquid density, evaporation, and air buoyancy. The total volume contained by the microtiter plate is divided by the number of filled wells. Thus, this procedure measures the average liquid delivery volume. This method is disadvantageous for calibrating multiple-orifice delivery devices because no information is provided on the amount delivered from an individual orifice.
In the photometric method, a sample holder having a transparent bottom surface receives the liquid volume to be determined. A beam of light passes through the bottom surface and the liquid and eventually to a detector. The amount of light absorbed, i.e. the absorbance, provides information on the depth of the liquid and thus, the volume, taking into account certain properties of components of the liquid, such as concentration and molar absorptivity (or extinction coefficient). For example, if the liquid volume contains a dye capable of absorbing the light, the amount of liquid present can be estimated by measuring the amount of light absorbed as it passes through the well. The more liquid dispensed into a given well, the deeper the column of liquid, and the more light absorbed. Spectrophotometers for measuring the absorbance of the liquid by this technique are well known and are commonly called microplate readers or microtiter plate readers, where the photometric method is used with specially constructed microtiter plates. In the current implementation, however, the photometric method fails to provide an adequate level of accuracy for many uses.
Another means of calibration is to dispense liquid that contains a diluted amount of a fluorophore, such as fluorescein, into a microtiter plate. A specialized plate reader measures the amount of fluorescence coming from each well of the microtiter plate. As with the photometric method, this method is generally not sufficiently accurate to satisfy the needs of many users. There are no stable or recognized standards for calibrating the sensitivity of such fluorescent readers, making it impossible for this method to be used alone to provide a quantitative result traceable to a national standard (e.g., as set by a recognized standards organization, such as ASTM).
In select embodiments, the present invention provides an easy to use, fast, accurate and precise method and apparatus for the calibration of liquid delivery devices. It provides results that are traceable to (within) national standards. The method is particularly useful for the calibration of small liquid volumes, such as in multichannel delivery devices. It allows semi-skilled laboratory technicians to practically and easily calibrate such equipment and on a schedule that provides for data integrity.
According to one aspect of the invention, a method is provided for calibrating a liquid volume. The method includes providing a sample solution including a first chromophore having an absorbance maximum at a first wavelength and a second chromophore having an absorbance maximum at a second wavelength. The difference between the first and second absorbance maxima is at least about 100 nm. The sample solution is exposed to electromagnetic radiation, and the absorbance by each chromophore is measured. A blank solution is also exposed to electromagnetic radiation, the blank solution being free of the first chromophore and including the second chromophore in a concentration equal to that in the sample solution. The absorbance of the blank solution is measured. A volume of the sample solution is determined based upon the measured absorbances of the blank solution and the sample solution.
According to another aspect of the invention, a liquid volume calibration system is provided including a spectrophotometer for emitting and detecting electromagnetic radiation. A multi-well plate is provided for containing a plurality of sample solutions and for exposing the solutions to the electromagnetic radiation. Each of the plurality of sample solutions includes a first chromophore having an absorbance maximum at a first wavelength and a second chromophore having an absorbance maximum at a second wavelength, the difference between the first and second absorbance maxima being at least 100 nm, and each sample solution having a unique concentration of at least the first chromophore. A separate blank solution is provided free of the first chromophore and including the second chromophore in a concentration equal to that in the sample solution.
According to another aspect of the invention, a system is provided including the plurality of sample solutions described in the immediately preceding paragraph, and a multi-well plate for containing the plurality of sample solutions and for exposing the solutions to electromagnetic radiation. Each well of the multi-well plate has a path length dimension provided to a level of error of no more than 0.5%. A separate blank solution is provided free of the first chromophore and including the second chromophore.
According to another aspect of the invention, a system is provided including a calibration plate for calibrating a first spectrophotometer with a second spectrophotometer, the calibration plate having multiple cells containing a first set of calibration solutions. The system further includes a second set of sample solutions each including a first chromophore having an absorbance maximum at a first wavelength and a second chromophore having an absorbance maximum at a second wavelength, the difference between the first and second absorbance maxima being at least one 100 nm. A multi-well plate is provided for containing a plurality of the sample solutions for use in the first spectrophotometer. A separate blank solution is provided free of the first chromophore and including the second chromophore in a concentration equal to that in the sample solution.
According to another aspect, a method is provided for determining a liquid volume. The method includes a step of providing a multi-well plate, providing a sample solution having an unknown volume and contained in a well of the multi-well plate, and maintaining a contact angle from about 80 to 100 degrees between a meniscus of the sample solution and the well, the contact angle being determined by concentrations of one or more of a chromophore, a salt, and a buffer in the sample solution. The sample solution is exposed to electromagnetic radiation and the absorbance of the chromophore is measured. The volume of the solution is determined based on the measured absorbance and the concentration of the chromophore.
In yet another embodiment, a computer-executable software code is stored on a computer-readable medium, the code including code for calculating a volume of a liquid sample solution based upon a photometric reading of absorbance, a concentration of a chromophore in the sample solution, a path length dimension of a sample holder in which the reading is made, and a quantification of a non-linearity from the Beer-Lambert law of the reading.